Particles

ABSTRACT

Particles comprising a branched polymer and either a block copolymer or a linear dendritic hybrid represent a category of useful materials. They may be used in for example drug delivery applications. They may be prepared by a method comprising the steps of: dissolving the branched polymer and block copolymer or linear dendritic hybrid, and optionally other component(s), in a solvent to form a solution; adding said solution to a different liquid; and removing said solvent to form a dispersion of co-precipitated particles.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a Continuation of U.S. Nonprovisional patentapplication Ser. No. 15/326,775, filed Jan. 17, 2017, which is the U.S.National Phase of International Patent Application Serial No.PCT/GB2015/052089, filed Jul. 17, 2015, which claims priority to GBApplication No. 1412841.7, filed Jul. 18, 2014. The entire disclosuresof the applications noted above are incorporated herein by reference.

The present invention relates to particles comprising organic polymers.These particles are useful in for example drug delivery applications.

We have previously disclosed various polymeric materials andtechnologies to produce molecular architectures which are useful invarious fields including biology and nanotechnology.

For example WO 2009/12220 discloses polymer-dendrimer hybrids, alsoknown as polydendrons, which comprise a branched vinyl polymer scaffoldcarrying dendrons: these polydendrons possess advantageousdendrimer-type properties in part due to their multiply branched nature,without the disadvantages of complex conventional dendrimer processes.

It is useful to be able to control the properties of such materialsincluding for example their size, size distribution, functionality,hydrophobicity/hydrophilicity, and behaviour in systems of use, e.g. inaqueous systems in the case of biological applications.

We have now developed a new category of useful materials.

From a first aspect the present invention provides particles comprisinga branched polymer and either a block copolymer or a linear dendritichybrid.

The branched polymer may be of various types. Two example types arebranched vinyl polymers and branched polyesters.

Branched vinyl polymers may be prepared by known methods, frommonofunctional vinyl monomers and difunctional vinyl monomers (branchingagents).

Branched polyesters may be prepared by for example ring openingpolymerization of monofunctional lactone monomers and difunctionallactone monomers (branching agents).

Block copolymers comprise blocks or segments each of which haverepeating units of the same type within each block and which aredifferent between block segments. The present invention has been foundto be particularly effective with diblock copolymers, i.e. copolymerswhich have two distinct blocks. Other block copolymers may be used, e.g.triblock copolymers.

Linear dendritic hybrids, also known as linear dendritic polymers,likewise comprise distinct segments, one of which is a linear polymerchain and another of which is a dendron. We have found that the presentinvention is particularly effective with a linear dendritic hybrid whichcomprises a homopolymer terminating in or initiated by a dendron.Nevertheless other types of linear dendritic hybrid (e.g. wherein thelinear polymer is itself a copolymer, for example a statisticalcopolymer, block copolymer or diblock copolymer) may be used in thepresent invention. Thus for example a linear dendritic hybrid maycomprise one block joined to another block which is then joined to adendron.

The particles may be nanoparticles, for example particles wherein atleast one dimension, or wherein the z-average diameter (also referred toas the average hydrodynamic diameter), is no greater than about 1000 nm.The particles may have z-average diameters less than 800 nm, or lessthan 500 nm, or less than 300 nm, e.g. around 50 to 300 nm, or 50 to 250nm, or 100 to 200 nm.

It is important to note that the particles of the present inventioncontain two types of polymeric material, i.e. a branched polymer andeither a block copolymer or a linear dendritic hybrid (and optionallyfurther materials as discussed below) but that these are not covalentlybonded to each other. In contrast, polydendrons, as disclosed in forexample WO 2009/12220, contain dendron units which are covalentlyattached to a branched vinyl polymer scaffold. In the present invention,the different polymeric materials are associated with each other withina particle structure, but are not covalently bonded to each other.Preferably most, or substantially all, or all, of the particles have thesame general structure. The regular way in which the parts associate,intermingle or assemble brings about reliable and reproducibleproperties.

The combination of polymeric structures in the present invention bringsabout particular advantages.

The first essential component, the branched polymer component, may begenerally hydrophobic and therefore useful for providing an environmentfor associating with, carrying, or encapsulating, a hydrophobiccompound, moiety or payload, for example a drug. Alternatively it mayhave hydrophilic properties. It may be responsive to pH or otherconditions or stimuli such that its properties, e.g. itshydrophobic/hydrophilic properties, its structure, and/or its behaviourin liquid phases, may vary depending on the environment.

The second essential component has at least two segments (differenthomopolymer or copolymer blocks in the case of block copolymers, or alinear polymer block plus a dendron in the case of linear dendritichybrids). This allows the resultant particles to be suitable for ortailorable for use in a variety of media and environments and for avariety of applications. It also allows one of the segments to act as astabilizer to stabilize the particles within liquid media, for examplein aqueous media, and optionally to provide salt stability to stabilizethe particles in a range of salt conditions and/or in a range ofphysiological conditions which might be encountered in living systems.

The block copolymer or linear dendritic hybrid may comprise ahydrophobic segment. This enables it to associate with a hydrophobicbranched polymer (and optionally with hydrophobic carried material). Thehydrophobic segment may comprise a vinyl polymer.

The block copolymer or linear dendritic hybrid may comprise ahydrophilic segment. This is useful in for example aqueous environmentsand enables the stabilisation of the nanoparticles. The hydrophilicsegment may comprise a polyether structure such as polyethylene glycol(PEG) or PEO, for example.

Of course, the block copolymer or linear dendritic hybrid may compriseboth a hydrophobic segment and a hydrophilic segment and thereby exhibitamphiphilic behaviour.

Thus we have developed particularly advantageous combinations where thebranched polymer is hydrophobic and forms a hydrophobic core and wherethe block copolymer or linear dendritic hybrid is amphiphilic such thatone segment thereof can associate with the hydrophobic core and anothersegment thereof can stabilise the particles in aqueous media. Theseexhibit considerable uniformity in terms of size, and stability inaqueous media and under varied salt conditions.

Nevertheless other combinations of types of polymer and consequentproperties are also useful in other scenarios, and are within the scopeof the present invention.

The branched polymer, block copolymer or linear dendritic hybrid maycomprise chemistry which is responsive to pH or other conditions orstimuli such that its properties, e.g. its hydrophobic/hydrophilicproperties, may vary depending on the environment.

The linear dendritic hybrid comprises a linear polymer chain and adendron. Said linear polymer chain may itself be a copolymer, e.g. astatistical copolymer or a block copolymer.

The particles may comprise a further material, for example a chemicalcompound, organic compound, hydrophobic compound, hydrophilic compound,drug, prodrug or therapeutically, diagnostically or biologically usefulmaterial. Indeed, one of the main uses of the present invention is indrug delivery.

The further materials, e.g. drugs or biologically active materials, maybe included as or within separate compounds within the particles, or maybe covalently bound to one of the polymeric structures, e.g. to thebranched polymer, block copolymer or linear dendritic hybrid

From a further aspect the particles are prepared by co-precipitation.

The method of preparation may comprise:

-   -   dissolving the branched polymer and either block copolymer or        linear dendritic hybrid, and optionally other component(s), in a        solvent to form a solution    -   adding said solution to a different liquid    -   removing the solvent, to form a dispersion of co-precipitated        particles within the liquid.

The solvent may be removed by for example allowing said solvent toevaporate or through dialysis.

The dispersion may be used for applications, e.g. therapeuticapplications, as it is, or alternatively the dispersion may beconcentrated or gelled, or the liquid may be removed to result in solidparticles.

The solvent in which the materials are dissolved may be an organicsolvent, suitably a solvent which is miscible with the liquid in whichprecipitation occurs, e.g. water-miscible solvents. For example thesolvent in which the materials are dissolved may be e.g. acetone,acetonitrile, dimethyl formamide, dimethyl sulfoxide, dioxane, ethanol,isopropyl alcohol, methanol, tetrahydrofuran and any combination andmixture of them or combinations comprising water.

The solvent in which the materials are dissolved may be one which ischosen because it evaporates relatively easily, e.g. one which has aboiling point of 80 degrees C. or less, or 70 degrees C. or less, or 60degrees C. or less.

The liquid in which the precipitation occurs may for example be water orother aqueous system, or other liquid in which the materials do notdissolve, or dissolve less well in comparison to the first solvent.

Various aqueous solutions can be used. Some examples include:

-   -   aqueous solutions of for example alkali metal/halogen salts        (e.g. NaCl, LiBr, KCl). at all concentrations (up to saturation)    -   aqueous solution within a pH range of 0 to 12, including acid        and base (BrØnsted-Lowry mineral and organic acids and bases)        solutions (e.g. aqueous solutions of e.g. HCl, H2SO4, acetic        acid, NaOH, or KOH)    -   surfactant (e.g. Sodium dodecyl sulphate, amphiphilic diblock        copolymers, TWEEN®, BRIJ®) solutions in water at all        concentrations (below and above critical micellar concentration)    -   biological media [e.g. transport buffer, bovine serum albumin        (BSA), dulbecco's modified eagle's medium (DMEM), Roswell park        memorial institute 1640 (RPMI 1640), foetal bovine serum (FBS)]        in aqueous systems, and mixtures of the same, at all suitable        concentrations.

The aqueous systems listed above provide some indication of the widerange of systems in which the present invention works, consistent withapplications in a variety of conditions including therapeuticapplications, where there is a need to endure varied and sometimesextreme physiological environments.

The liquid in which precipitation occurs may be a mixture of water andorganic solvent e.g. acetone/water mixture, THF/water, MeOH/water,EtOH/water, or IPA/water. The amount of water in such mixtures mayoptionally be 2 parts water by volume to 1 part organic solvent byvolume, or greater than 2 parts water (e.g. greater than 3 parts water,e.g. greater than 4 parts water) by volume to 1 part organic solvent byvolume.

One of the inventive points of distinction of the present invention overthe prior art is that the present invention involves theco-precipitation of two specific types of polymer at the same time fromorganic solvent into water. Whereas precipitation (includingnanoprecipitation) of organic polymers has previously been disclosed invarious documents and has been carried out with varying degrees ofsuccess (see, for example, Slater et al, Soft Matter 2012, 8,9816-9827), this has not been in relation to a combination of polymertypes as defined in the present invention. We are first to recognize,and demonstrate, the benefits of such co-precipitation.

Optionally, the co-precipitation may be carried out multiple times.Thus, after the formation of a dispersion of co-precipitated particleswithin the liquid (and the subsequent removal of organic solvent), thefollowing method steps may additionally be carried out, one or moretimes:

-   -   adding further solution (of the branched polymer and either the        block copolymer or the linear dendritic hybrid, and optionally        the other component(s), in the solvent) to the liquid    -   removing the solvent.

We have found that, by carrying out multiple co-precipitation, it ispossible to increase the concentration of particles within the liquidwhilst substantially maintaining the particle size, PdI and stability.In other words, multiple nanoprecipitation can be used to provide moreof the same type of nanoparticles per unit volume, whereas it might havebeen expected that multiple nanoprecipitation would result insignificantly larger particles, different dispersity characteristics, orinstability. This is particularly advantageous in facilitating drugdelivery where an enhanced payload can result in practical therapeuticadvantages and cost benefits.

The Branched Polymer Component

The branched polymer is an essential component of the particles of thepresent invention.

The branched polymer is non-gelled and processable and optionally of lowviscosity. It is soluble in organic solvents, as described above inrelation to a suitable method of forming the particles by precipitationfrom such solvents into a different medium. It can be contrasted withpolymer structures which are insoluble and/or exhibit high viscosity,such as extensively crosslinked insoluble polymer networks, highmolecular weight linear polymers, or microgels.

Branched Vinyl Polymers

The branched polymer may be a branched vinyl polymer. This can be madeby, but is not limited to being made by, living polymerization,controlled polymerization or conventional chain-growth polymerizationtechniques such as free radical polymerisation. Several types of livingand controlled polymerization are known in the art and suitable for usein the present invention. A preferred type of living polymerization isAtom Transfer Radical Polymerization (ATRP); however other techniquessuch as Reversible Addition-Fragmentation chain-Transfer (RAFT) andNitroxide Mediated Polymerisation (NMP) or conventional free-radicalpolymerization controlled by the deliberate addition of chain-transferagents are also suitable syntheses.

The skilled person is aware of techniques to provide branched butnon-gelled vinyl polymers. For example, suitable procedures aredescribed in N. O'Brien, A. McKee, D. C. Sherrington, A. T. Slark and A.Titterton, Polymer 2000, 41, 6027-6031; T. He, D. J. Adams, M. F.Butler, C. T. Yeoh, A. I. Cooper and S. P. Rannard, Angew. Chem. Int.Ed. 2007, 46, 9243-9247; V. Bütün, I. Bannister, N. C. Billingham, D. C.Sherrington and S. P. Armes, Macromolecules 2005, 38, 4977-4982; I.Bannister, N. C. Billingham, S. P. Armes, S. P. Rannard and P. Findlay,Macromolecules 2006, 39, 7483-7492; and R. A. Slater, T. O McDonald, D.J. Adams, E. R. Draper, J. V. M. Weaver and S P Rannard, Soft Matter2012, 8, 9816-9827. The non-gelled and soluble products of the presentinvention are different to materials disclosed in L. A. Connal, R.Vestberg, C J. Hawker and G. G. Qiao, Macromolecules 2007, 40, 7855-7863which are known to comprise multiple cross-linking in a gelled network.

The polymerization of each vinyl polymer chain starts at an initiator.Polymerization of monofunctional vinyl monomers leads to linear polymerchains. Copolymerization with difunctional vinyl monomers leads tobranching between the chains. In order to control branching and preventgelation there should be less than one effective brancher (difunctionalvinyl monomer) per chain. Under certain conditions, this can be achievedby using a molar ratio of brancher to initiator of less than one: thisassumes that the monomer (i.e. the monofunctional vinyl monomer) and thebrancher (i.e. the difunctional vinyl monomer) have the same reactivity,that there is no intramolecular reaction, that the two functionalitiesof the brancher have the same or similar reactivity, and that reactivityremains the same even after part-reaction. Of course, the systems andconditions may be different, but the skilled person understands how tocontrol the reaction and determine without undue experimentation how anon-gelled structure may be achieved. For example, under diluteconditions some branchers form intramolecular cycles which limit thenumber of branchers that branch between chains even if the molar ratioof brancher to initiator (i.e. polymer chain) is higher than 1:1 in thereaction.

Initiators and other reagents used in the polymerisation process are asknown in the art. For example, in ATRP, convenient and effectiveinitiators include alkyl halides (e.g. alkyl bromides) and inconventional free radical polymerisation, effective initiators includeazo compounds

Other Branched Polymers

Other suitable types of branched polymers include branched polyesters.These may be prepared by for example ring opening polymerization ofmonofunctional lactone monomers and difunctional lactone monomers(branching agents). Ring opening polymerization methods and materialsare known in the art, for example from Nguyen et al., Polym Chem 2014,5, 2997-3008.

Amount of Branched Polymer in Relation to Linear Polymer and OtherOptional Components

The amount of branched polymer (e.g. branched vinyl polymer) mayoptionally be no greater than 95 wt %, or no greater than 75 wt %, or nogreater than 50 wt %, or no greater than 10 wt %, or no greater than 1wt %. In this context wt % denotes the amount of branched vinyl polymeras a percentage of the total mass of solid material in the particles.The amount of branched polymer may optionally be at least 0.1 wt %, orat least 0.5 wt %. For example the amount of branched polymer may bebetween about 0.1 wt % and 95 wt %, e.g. between 1 wt % and 10 wt %.

Surprisingly we have found that relatively low amounts of branchedpolymer (optionally around 1-10 wt % in some cases) are effective inbeing able to direct the nanoprecipitation, and in allowing theformation of regular particles of narrow dispersity. Thus, in someapplications this can bring a benefit in allowing the amount of othercomponent(s) (i.e. the block copolymer component or the linear dendritichybrid, plus any other components e.g. carried materials) to bemaximised.

The Block Copolymer Component

The block copolymer component can comprise various types of polymerincluding for example vinyl polymers, polyethers (e.g. PEO or PPO),polyesters (e.g. polycaprolactone or polylactic acid), polyurethanes,polyamides, or polycarbonates.

There are (at least) two distinct blocks. The type of polymer formingeach block may be the same or different, though of course if the type ofpolymer is the same then for the blocks to differ the monomers, ormixture of monomers, must differ.

For example, the block copolymer may comprise two blocks each of whichis a vinyl polymer chain, wherein the vinyl monomer making up one blockdiffers from the vinyl monomer making up the other block.

Alternatively the type of polymer forming each block may be different.For example the block copolymer may comprise one block which is a vinylpolymer chain and a second block which is a polyether chain or apolyester chain.

One convenient known way of preparing a block copolymer wherein oneblock is a vinyl chain is to use a macroinitiator in a vinylpolymerisation process wherein the macroinitiator comprises the other oranother block. For example, the macroinitiator may be a bromideinitiator comprising a PEO or other polymer chain. In a furthervariation, said initiator may be bifunctional and may therefore allowthe preparation of a triblock copolymer (for example being an A-B-Ablock copolymer where A is a vinyl, often hydrophobic, block, and B is aPEO, hydrophilic, block).

The molecular mass of the PEO may for example be greater than 1,000g/mol, for example between 1,000 and 10,000 g/mol; for example PEG 2K orPEG 5K.

Another example is to use a polymer (e.g. a hydrophilic polymer) bearinga hydroxyl group as a macroinitiator for ring opening polymerisation(e.g. of cyclic esters, e.g. □-caprolactone or other cyclic structures).Known ring-opening polymerisation techniques may be used.

Other ways to synthesise diblock copolymers include for example one-potmethods in which one monomer (e.g. a vinyl monomer) is polymerised first(generally a hydrophilic block first, but not necessarily) and then,when this first reaction has reached the desired monomer conversion, andwithout any purification of the first block, the second monomer (whichmay for example lead to a hydrophobic block if the first monomer leadsto a hydrophilic block) may be added in situ. In each case, the monomersadded may be a mixture of monomers leading to a statisticalcopolymerisation within each block. Other known methods of preparingblock copolymers may be used.

The Linear Dendritic Hybrid

The linear dendritic hybrid comprises a polymer chain and a dendron. Thedendron is covalently attached to the polymer chain. The polymer chaincan comprise various types of polymer including vinyl polymers,polyesters, polyamides, polycarbonates, polyurethanes, or polyethers(e.g. PEO or PPO).

Linear dendritic hybrids are known from various documents, e.g. F. Wurm,H. Frey, Prog. Polym. Sci. 2011, 36, 1-52.

The dendron may be incorporated in various ways. One possible methoduses dendrons as macromolecular initiators in for example vinylpolymerisation. This allows the formation of for example lineardendritic hybrids having a vinyl polymer chain with a dendron at the endof the chain. Dendrons may also be used as chain transfer agents toregulate and initiate a chain growth polymerisation.

In order to be able to initiate polymerization, the dendrons must bearsuitable reactive functionality. For example, in ATRP, dendrons whichcarry halides (e.g. bromides) at their focal points can act asinitiators. In this scenario, propagation starts at the apex, or focalpoint, of the dendron “wedge”. The skilled person is well aware of thetypes of components and reagents which are used in polymerisationsincluding ATRP and other living or controlled polymerizations andconventional free radical polymerisation, and hence the type offunctionality which must be present on or introduced to dendrons forthem to act as initiators or chain transfer agents.

For example, one possible way of introducing bromo groups to dendrons isto functionalize dendron alcohols with alpha-bromoisobutyryl bromide.There are however many other ways of functionalizing dendrons so thatthey can act as initiators and chain transfer agents, and other types offunctionality which will initiate polymerization. The concept of adendron initiator is applicable to all suitable types of polymerizationand the functionality can be varied as necessary.

Another method of incorporating dendron comprises ring openingpolymerisation e.g. of cyclic esters, using dendrons with suitablefunctional groups (e.g. —OH groups).

A further method uses dendrons as chain transfer agents forpolymerisation with reversible addition-fragmentation chain transfer(RAFT) or conventional free radical polymerisation.

Other possible methods include post-functionalisation of polymers viachain-end/dendron coupling, for example through the use of a thiol groupat the focal point.

Other methods known in the art for the incorporation of dendrons intolinear dendritic hybrids may be used.

There is no particular limitation regarding the type of dendron that canbe used, or the chemistry used to prepare the dendrons. In somescenarios it is desirable to have particular groups present at thesurface (i.e. at the tips of the “branches” of the dendron), and thesemay be incorporated during the synthesis of the dendron. The dendronsare preferably non-vinyl.

Any suitable coupling chemistry may be used to build up the dendrons. Inone example, amines and alcohols may be coupled together, for exampleusing carbonyldiimidazole. This is, however, merely one example andnumerous other coupling methods, such as Michael addition chemistry orwell known esterification techniques, are possible.

The dendrons may comprise various moieties including for example amines(e.g. dendrons which are branched at tertiary amine centres, or dendronswhich terminate at NMe₂ groups), hydroxyl groups, acids, carboxylgroups, or PEG groups.

The branched polymer may optionally contain alcohol groups.

The branched polymer may optionally contain amine groups.

The branched polymer may optionally contain carboxyl groups.

The block copolymer may optionally contain alcohol groups.

The block copolymer may optionally contain amine groups.

The block copolymer may optionally contain carboxyl groups.

The polymer chain of the linear dendritic hybrid may optionally containalcohol groups.

The polymer chain of the linear dendritic hybrid may optionally containamine groups.

The polymer chain of the linear dendritic hybrid may optionally containcarboxyl groups.

EXAMPLES, FIGURES AND EXPERIMENTAL DETAILS

The present invention will now be described in further non-limitingdetail, by way of example, with reference to the figures in which:

FIG. 1 shows a schematic representation of a branched vinyl polymer,diblock copolymers, and a particle of the present invention containingthe branched vinyl polymer and diblock copolymers, and also somemonomers which may be used in the preparation of the polymers;

FIG. 2 shows a schematic representation of one possible co-precipitationprocedure in accordance with the present invention;

FIG. 3 shows SEM images of some nanoparticles in accordance with thepresent invention;

FIGS. 4 and 5 shows the stability of particles of the present inventionto salt solution and over time;

FIG. 6 shows an SEM image of nanoparticles in accordance with thepresent invention;

FIGS. 7 to 12 show particle size distributions of drug-loaded particles;

FIG. 13 shows a schematic representation of a branched polymer, lineardendritic hybrid, and a nanoparticle of the present invention containingboth, in two different architectures;

FIGS. 14 to 18 show some combinations of branched polymers and lineardendritic hybrids and their properties;

FIG. 19 shows an SEM image of some nanoparticles; and

FIG. 20 shows the effect on cell viability of some nanoparticles of thepresent invention.

PARTICLES CONTAINING BRANCHED POLYMERS AND BLOCK COPOLYMERS

One group of particles in accordance with the present invention arethose which contain branched polymers and block copolymers.

FIG. 1 shows an overview of some components used. A branched vinylpolymer (top left) contains linear chains formed from vinyl monomerswhich are branched by use of a brancher. A wide variety of vinylmonomers may be used, some examples of which are shown (bottom left).

The vinyl monomers shown in the bottom left of FIG. 1, and theirabbreviations as used herein, are as follows:

-   -   HPMA: hydroxypropyl methacrylate. 2-hydroxypropyl methacrylate        is shown, though the invention can utilize not only this isomer        alone but optionally a commercially available mixture of this        isomer and 2-hydroxyisopropyl methacrylate or the latter alone.    -   nBuMA: n-butyl methacrylate    -   Styrene    -   tBuMA: t-butyl methacrylate    -   DEAEMA: N,N-diethylaminoethyl methacrylate, a monomer which is        hydrophilic in comparison to the above-mentioned monomers, and        pH-responsive.

As indicated in FIG. 1, combinations of monomers may be used e.g. HPMAand nBuMA, or HPMA and tBuMA.

Other materials used include the following, also shown in FIG. 1:

-   -   EBIB: ethyl alpha-bromoisobutyrate, an initiator commonly used        to initiate polymerisation    -   EGDMA: ethylene glycol dimethacrylate, a suitable brancher    -   PEG 2K or PEG 5K: polyethylene oxide of size 2K or 5K (although        of course other sizes are possible), which is        bromo-functionalised so that it can act as a macroinitiator for        vinyl polymerisation. Because of the number of ethylene oxide        groups present in the polyethylene oxide (PEO), PEG 2K is also        referred to as PEO₄₅ and PEG 5K is also referred to as PEO₁₁₄.

Other monomers, branchers, initiators and other materials can also beused including:

-   -   BDME, 1,4-butanediol di(methacryloyloxy)-ethyl ether, a pH        responsive brancher

Diblock copolymers of varying lengths are shown (top middle): these canfor example have one block formed from a vinyl monomer selected fromthose shown, e.g. HPMA, and a second block of different chemistry e.g.polyethylene oxide. One possible preparative procedure usesbromo-functionalised polyethylene oxide (e.g. PEG 2K or 5K) (FIG. 1,bottom right) as a macroinitiator for vinyl polymerisation. Theresultant amphiphilic diblock copolymer can be co-precipitated with thebranched vinyl polymer to form a particle which is schematicallyillustrated top right. In this example the hydrophilic polyethyleneoxide chains of the block copolymer are shown on the outside of theparticle, as would be the case in aqueous systems.

As shown in FIG. 2, the co-precipitation may for example be carried outby adding both polymers, in suitable organic solvent, to water, and thenallowing the organic solvent to evaporate.

Some examples of particle size (dynamic light scattering size) andpolydisersity (PDI) values for co-precipitates of branched vinyl polymer(poly-HPMA-EGDMA) with amphiphilic diblock copolymer (one block beingPEG 5K or PEG 2K, and the other being HPMA₄₀, HPMA₈₀, or HPMA₁₂₀) invarious ratios are shown in the following table:

Branched polymer:diblock PEG2K PEG2K PEG2K PEG5K PEG5K PEG5K polymerHPMA40 HPMA80 HPMA120 HPMA40 HPMA80 HPMA120 90/10 Z-ave (d nm) 113 114120 111 112 132 PDI 0.067 0.045 0.066 0.055 0.045 0.059 80/20 Z-ave (dnm) 94 127 111 103 106 127 PDI 0.123 0.061 0.033 0.152 0.118 0.043 70/30Z-ave (d nm) 106 115 108 121 106 113 PDI 0.029 0.053 0.037 0.061 0.1240.069 60/40 Z-ave (d nm) 115 111 103 107 103 117 PDI 0.047 0.037 0.0690.028 0.065 0.059 50/50 Z-ave (d nm) 167 124 226 154 141 135 PDI 0.0240.055 0.035 0.048 0.063 0.051

It can be seen that, in these examples, the sizes (z-average diameters)of the co-nanoprecipitated particles range from 90 to 230 nm, and thatthere are narrow PDI values throughout showing their consistency anduniformity.

SEM shows the sizes and spherical nature of the nanoparticles. FIG. 3shows some examples: co-precipitates of branched vinyl polymer(poly-HPMA-EGDMA) with PEG 5K-HPMA₁₂₀ in the ratios 80:20 (top) and90:10 (middle) and with PEG 2K-HPMA₁₂₀ in the ratio 50:50 (bottom).

FIG. 4 shows the stability of various particles to salt solution (0.5MNaCl). “Blank” denotes before salt is added, “Instant” immediatelyafter, and 1, 7 and 21 the number of days afterwards. The particlesexhibit considerable salt stability, which is important for use inphysiological environments. Previous examples of particles formed solelyfrom branched vinyl polymers alone have been shown to be unstable in thepresence of salts (Slater et al, Soft Matter 2012, 8, 9816-9827).

The following example provides more details of some nanoparticlescontaining hydrophobic cores and amphiphilic diblock stabilisers:

Example 1: Nanoparticles Containing Branched Hydrophobic Core Polymerand Linear Amphiphilic Diblock Copolymer Preparation of Polymers

ATRP was used for the synthesis of linear PEO₄₅-p(HPMA₁₂₀) and thebranched statistical copolymer of EGDMA and HPMA p(HPMA₅₀-EGDMA_(0.9)).Polymers were analysed by triple detection gel permeation chromatography(GPC) and ¹H NMR. Monomer to polymer conversion was monitored by ¹H NMRusing anisole as an internal reference.

The linear amphiphilic block copolymer was synthesised during a one stepATRP reaction in methanol at 30° C. For introduction of the hydrophilicblock a PEO₄₅-Br macroinitiator (2 kDa) was added accordingly to HPMAalong with the catalytic system Cu(I)Cl/2,2′-Bipyridine (Bpy) in thefollowing ratio [Macroinitiator]:[Monomer]:[Cu(I)Cl]:[Bpy]=1:120:1:2.The targeted number average degree of polymerisation (DP_(HPMA)) was 120HPMA monomer units and GPC confirmed a number average DP_(HPMA)=127monomer units (M_(n)=20300 Da) with a narrow dispersity (Ð=1.23).

The branched statistical copolymerisation of HPMA and EGDMA wasinitiated with ethyl 2-bromo isobutyrate (EBIB) using the catalyticsystem previously described in the following ratio[Macroinitiator]:[EGDMA]:[Monomer]:[Cu(I)Cl]:[Bpy]=1:0.9:50:1:2. Themolar ratio of [EGDMA]/[EBIB]<1 is a crucial parameter in order to havecontrol over the branching reaction and avoid gelation. As expected, thepresence of EGDMA dramatically increases the polymer molecular weight(for example, M_(w)=295,000 Da) and dispersity (Ð=7.56) in contrast tothe linear amphiphilic polymer which displayed a monomodal narrowmolecular weight distribution. In addition, ¹H NMR analysis of bothlinear PEO₄₅-p(HPMA₁₂₀) and branched p(HPMA₅₀-EGDMA_(0.9)) showed highmonomer conversion (>98%).

Nanoprecipitation and Co-Nanoprecipitation

Polymeric nanoparticles were prepared by nanoprecipitation whichcorresponds to a solvent switch through a rapid precipitation into water(ambient temperature). It is hypothesised that during the association ofthe p(HPMA₅₀-EGDMA_(0.9)) hydrophobic branched core, the linear HPMAchains from the diblock copolymer also become incorporated into thehydrophobic core allowing the PEO₄₅ (2 kDa) chains to be present at thesurface of the resulting particles and prevent aggregation by stericstabilisation. Varying weight fractions ofp(HPMA₅₀-EGDMA_(0.9))_(x):PEO₄₅-p(HPMA₁₂₀)_(y) (x:y) were dissolved inacetone at a total concentration of 5 mg mL⁻¹ for six hours to ensurecomplete solubilisation. Rapid precipitation of 1 mL of the polymersolution into 5 mL of water gave a final nanoparticle concentration of 1mg mL⁻¹ after complete acetone evaporation. The nanoparticle dispersionswere analysed by dynamic light scattering (DLS) and scanning electronmicroscopy (SEM). The resulting z-average diameters (nm), polydispersityindexes (PDI), zeta potentials (mV) and number average diameters (nm)are collected in the following table.

TABLE DLS and SEM characterisation of nanoparticles obtained byco-nanoprecipitation of p(HPMA₅₀-EGDMA_(0.9)), PEO₄₅-p(HPMA₁₂₀) andp(HPMA₅₀-EGDMA_(0.9))_(x):PEO₄₅- p(HPMA₁₂₀)_(y)(x:y) acetone solutionsinto water. DLS DLS SEM Z- Number Number average Diameter Diameter ZetaRatio Diameter Average Average Potential Entry Sample (%) (d · nm) PDI(d · nm) (d · nm) (mV) 1 p(HPMA₅₀-EGDMA) 100 148 0.08 112 ± 6  117 −40.52 PEO₄₅-p(HPMA₁₂₀) 100 296 0.135 188 ± 80 190 −17.5 3p(HPMA₅₀-EGDMA):PEO₄₅- 90:10 107 0.075 84 ± 2 81 −19.3 p(HPMA₁₂₀) 4p(HPMA₅₀-EGDMA):PEO₄₅- 80:20 126 0.046 99 ± 4 105 −24.4 p(HPMA₁₂₀) 5p(HPMA₅₀-EGDMA):PEO₄₅- 70:30 116 0.067 93 ± 3 117 −20.6 p(HPMA₁₂₀) 6p(HPMA₅₀-EGDMA):PEO₄₅- 60:40 119 0.107 91 ± 1 88 −21.5 p(HPMA₁₂₀) 7p(HPMA₅₀-EGDMA):PEO₄₅- 50:50 174 0.093 137 ± 3  110 −19.7 p(HPMA₁₂₀)

Self assembly of the branched p(HPMA₅₀-EGDMA_(0.9)) and amphiphilicPEO₄₅-p(HPMA₁₂₀) polymers during co-nanoprecipitation from acetone intoH₂O produced well defined particles indicated by the low PDI values(0.046-0.107) and size homogeneity respectively obtained from DLSmeasurements and SEM observations.

The co-nanoprecipitated particles exhibited zeta potential valuesranging between −19 mV and −25 mV (Table 1, entries 3-7).

Salt Stability

The salt stability of the co-precipitated particles was demonstrated byadding aliquots (20 μL) of an aqueous 0.5M NaCl salt solution to 1 mL ofthe nanoprecipitated dispersions. Z-average diameters and PDI weremeasured during a period of 0-21 days.

Co-nanoprecipitated particles demonstrated excellent salt stability over21 days after 20 μL addition, and maintainance of narrow polydispersity.Nanoparticles containing only branched vinyl polymer crashed out ofsolution immediately on NaCl addition whereas nanoparticles containingboth branched vinyl polymer and diblock amphiphiic copolymer remained asa stable nanoparticle dispersion. SEM analysis of the nanoparticles (0.1mg mL⁻¹) showed the spherical nature of the particles andco-nanoprecipitates made up of groups of particles.

FIG. 5 shows how the z-average diameter and PDI vary on addition of NaCl(20 μL 0.5M NaCl) to a 1 mg mL⁻¹ aqueous dispersion ofp(HPMA₅₀-EGDMA_(0.9)):PEO₄₅-p(HPMA₁₂₀) 60:40. The vertical axis denotesintensity (percent) and the horizontal axis shows z-average diameter (d,nm). Solid line: before addition. Dashed line: immediately after NaCladdition. Dotted line: 21 days after NaCl addition.

FIG. 6 shows a scanning electron microscopy image ofp(HPMA₅₀-EGDMA_(0.9)):PEO₄₅-p(HPMA₁₂₀) 60:40; 1 mg mL⁻¹.

Conclusions from Example 1

Example 1 shows that the addition of an amphiphilic diblock ‘stabiliser’(PEO 2000 Da) to a branched hydrophobic polymer at low concentrationsfollowed by co-nanoprecipitation can form particles with desirablez-average diameters and very narrow PDI values. A range ofco-nanoprecipitated particles can be prepared. The co-nanoprecipitatedparticles offer enhanced stability due to the introduction of a stericstabiliser. This relies upon the formation of an outer layer of materialthat prevents particles coming into close proximity.

Loading of HIV Antivirals

The use of the nanoparticles to load various drugs including thefollowing HIV antiretrovirals was investigated:

Drugs could be encapsulated inside the polymer particles in a reliableand reproducible manner, allowing particles to be produced with verynarrow particle size distributions.

The use of branched vinyl polymers with a blend of two monomers [HPMAplus either tert-butyl methacrylate (tBMA) or n-butyl methacrylate(nBMA)] in the hydrophobic core was found to be particularly effectivein allowing high drug loadings to be achieved. Good results were alsoobtained when nBMA was used as the only monofunctional monomer in thebranched vinyl polymer.

The results are shown in FIGS. 7 to 12 as follows:

Branched polymer (EGDMA used as FIG. Drug and loading brancher in eachcase) Z-ave PdI 7 Ritonavir 20 wt % tBMA-HPMA 123 nm 0.062 8 Ritonavir20 wt % nBMA-HPMA 141 nm 0.072 9 Lopinavir 15 wt % tBMA-HPMA 166 nm0.060 10 Lopinavir 15 wt % nBMA-HPMA 137 nm 0.063 11 Efavirenz 15 wt %tBMA-HPMA 131 nm 0.037 12 Efavirenz 15 wt % nBMA-HPMA 117 nm 0.131

The block copolymer utilised to form particles shown in FIGS. 7-12 wasPEG 5K-HPMA₁₂₀ and the ratio of branched polymer to linear polymer was50:50.

The drug loading could be increased by further tailoring the polymerchemistry to allow drug loadings of 25 wt % to be achieved forEfavirenz. The drug-loaded particles were stable extended periods oftime. Further experiments investigated the loading of different drugtypes and details of these (and also of polymer synthesis andco-nanoprecipitation in this context) are as follows.

Typical Nanoparticle Preparation Including 10 wt % Loading forEfavirenz, Ritonavir and Lopinavir

During a typical nanoparticle preparation, 5.5 mL of a 1 mg/mL acetonesolution of efavirenz, ritonavir or lopinavir is added to a vial andleft to evaporate overnight. To this vial 25 mg of the branched polymerand 25 mg of the polymer diblock were added and dissolved in 10 mL ofacetone during 6-8 hours to ensure complete solubilisation. 1 mL of the5 mg/mL solution of polymers and dissolved drug was added to 5 mL ofstirring distilled water (500 rpm) and left for 24 hours for completeacetone evaporation (final concentration of polymer 1 mg/mL).

Anti-Cancer Drugs

Other examples of drugs which can be incorporated are anti-cancer drugs.

Irinotecan is a hydrophobic anticancer drug and SN-38 is a hydrophobicanticancer active metabolite of irinotecan.

Irinotecan in particular was effectively encapsulated at 10 wt % or 15wt % into co-nanoprecipitated particles with low PdI values, for examplewhere the branched polymer comprised nBMA or tBMA-HPMA and theamphiphilic polymer comprised PEG5K-HPMA120.

Irinotecan (Typical 10 wt % Loading)

During a typical nanoparticle preparation 5.5 mL of a 1 mg/mL acetonesolution of irinotecan was added to a vial and left to evaporateovernight. To this vial, 25 mg of the branched polymer and 25 mg of thepolymer diblock were added and dissolved in 10 mL of acetone during 6-8hours to ensure complete solubilisation. 1 mL of the 5 mg/mL solution ofpolymers and dissolved drug was added to 5 mL of stirring water (500rpm) and left for 24 hours for complete acetone evaporation.

SN-38 (Typical 2 wt % Loading)

During a typical nanoparticle preparation 1.03 mL of a 1 mg/mLTHF/Acetonitrile (50:50) solution of SN-38 was added to a vial and leftto evaporate overnight. To this vial 25 mg of the branched polymer coreand 25 mg of the polymer diblock were added and dissolved in 10 mL ofacetone during 6-8 hours to ensure complete solubilisation. 1 mL of the5 mg/mL solution of polymers and dissolved drug was added to 5 mL ofstirring water (500 rpm) and left for 24 hours for complete acetoneevaporation.

Preparation of SN-38 Nanoparticles by DMSO Dialysis

During a typical nanoparticle preparation by dialysis, 2.65 mL of a 1mg/mL DMSO solution of SN-38, 25 mg of the branched polymer core and 25mg of the polymer diblock were added and dissolved in 7.35 mL of DMSOduring 6-8 hours to ensure complete solubilisation. 1 mL of the 5 mg/mLsolution of polymers was added to a dialysis bag with a molecular weightcut off (MWCO) of 2000 g/mol and left to dialyse in distilled water over4 days (changing the water every 4 hours)

ATRP Polymerisation—Formation of the Branched Polymer Corep(HPMA₅₀-EGDMA_(0.9))

The targeted number average degree of polymerisation (DP_(n)) was 50repeat units. During a typical ATRP synthesis, EBIB initiator (0.14 g,0.69 mmol 1 eq.) and HPMA (5 g, 34.68 mmol 50 eq.) were added to around-bottomed flask equipped with a nitrogen inlet/outlet and a stirrerbar. Methanol was added (50 wt/wt %, based on HPMA) and the solution wasstirred vigorously under nitrogen for 10-15 minutes. The branching agentEGDMA (0.12 g, 0.62 mmol 0.9 eq. to EBIB initiator), copper catalystCu(I)Cl (0.069 g, 0.69 mmol 1 eq.) and bpy (0.22 g, 1.39 mmol 2 eq.)were added to the flask and the temperature was fixed at 30° C. Thereaction was monitored by ¹H NMR spectroscopy and terminated withmethanol when the HPMA monomer had reached >99% conversion. The polymerwas purified using Dowex Marathon exchange beads (˜12 g) to removeexcess copper catalyst followed by passing the sample through a basicalumina column. Excess THF was removed under vacuum to concentrate thesample before precipitation into cold hexane. The resulting polymer wasconfirmed by ¹H NMR in MeOD, triple detection GPC with an eluent of THF.

The polymerisation was carried out for all other monomers:

nBMA p(nBMA₅₀-EGDMA_(0.9)), tBMA p(tBMA₅₀-EGDMA_(0.9)), HPMA-nBMAp(HPMA₂₅-nBMA₂₅-EGDMA_(0.9)) and HPMA-tBMA p(HPMA₂₅-tBMA₂₅-EGDMA_(0.9))Example Synthesis of Poly(Ethylene Glycol) Mono-Functional ATRPMacro-Initiator (PEO_(x)-Br) (when x=45, the Macroinitiator is Referredto Herein as PEG 2K)

During a typical synthesis, PEO₄₅-OH (30 g, 15 mmol, 1 eq.) wasdissolved in 100 mL of toluene in the presence of triethylamine (2.275g, 22.5 mmol, 1.5 eq.) and 4-dimethylaminopyridine (0.092 g, 0.75 mmol0.05 eq.) in a two necked round-bottomed flask fitted with an additionfunnel, a nitrogen inlet/outlet and a stirrer bar.2-bromo-2-methylpropionyl bromide (5.175 g, 22.5 mmol, 1.5 eq.) dilutedwith 25 mL of toluene was placed in the addition funnel. The reactor wasput under stirring, cooled at about 0° C. in an ice bath and the2-bromo-2-methylpropionyl bromide solution was added slowly over aperiod of 20-30 min After the addition was completed, the reactor wasallowed to reach room temperature and was left to stir for 24 hours. Theformation of a white precipitate (triethylamine salt) indicated theprogress of the reaction. Then, the reaction medium was warmed up in awater bath at about 50° C., filtered and concentrated on the rotaryevaporator. The resulting product was diluted in acetone and purified byprecipitation in petroleum ether. The last step was repeated once andthe product was finally dried under vacuum at 40° C. for 24 hours. Theresulting macro-initiator was recovered with 70% yield and its structurewas confirmed by ¹H NMR in D₂O, triple detection GPC with an eluent ofDMF and MALDI-TOF mass spectrometry.

PEG 5K

Same synthesis for PEO-5K initiator—PEO₁₁₄-OH was used rather thanPEO₄₅-OH.

ATRP Polymerisation—Synthesis of Linear PEO₄₅-P(HPMA₁₂₀)

The targeted number average degree of polymerisation (DP_(n)) was 120repeat units. In a typical ATRP synthesis, PEO₄₅-Br macroinitiator (0.62g, 0.29 mmol 1 eq.) and HPMA (5 g, 34.68 mmol 120 eq.) were added to around-bottomed flask equipped with a nitrogen inlet/outlet and a stirrerbar. Methanol was added (33.5 w/v %, based on HPMA) and the solution wasstirred vigorously under nitrogen for 10-15 minutes. The copper catalystCu(I)Cl (0.029 g, 0.29 mmol 1 eq.) and bpy (0.09 g, 0.58 mmol 2 eq.)were added to the flask and the temperature was fixed at 30° C. Thereaction was monitored by ¹H NMR spectroscopy and terminated withmethanol when the HPMA monomer had reached 99% conversion. The polymerwas purified using Dowex Marathon exchange beads (˜12 g) to removeexcess copper catalyst followed by passing the sample through a basicalumina column. Excess THF was removed under vacuum to concentrate thesample before precipitation into petroleum ether 30/40. The resultingpolymer was confirmed by ¹H NMR in d₆-DMSO, triple detection GPC with aneluent of DMF.

The procedure described above was used for all other number averagedegree of polymerisations for HPMA and the ATRP of PEOSK-nBMA120.

Other Drugs and Other Drug Incorporation Methods

The present invention is also compatible with numerous other drugs andalso with other methods of incorporating drugs including not justencapsulation as described previously but also chemical bonding,sometimes referred to as conjugation, either to the branched polymer orthe linear polymer or both components of the particle.

In this context Ibuprofen was used as a model drug. Free ibuprofen wasencapsulated, and it was also bonded via its acid functionality toproduce a prodrug model.

Ibuprofen Work—Prodrug Model Synthesis of the Ibuprofen (IBU) ModifiedHPMA (IbuPMA)

During a typical synthesis HPMA (1.5 g, 10.40 mmol 1 eq.), Ibuprofen(2.79 g, 13.53 mmol 1.3 eq), DMAP (0.64 g, 5.5 mmol, 0.5 eq) and DCC(2.79 g, 13.53 mmol, 1.3 eq) were dissolved in 40 mL of THF in a roundbottom flask and stirred at ambient temperature for 24 hours. The DCUsalt was filtered and washed with THF followed by rotary evaporation.DCM (100 mL) was added and washed with 1M sodium bisulfate solution toremove excess DCU, then dried over MgSO4, concentrated in vacuo andstored at 0° C.

ATRP Polymerisation—Incorporated IBU Monomer: Targeted Total DP80

Composition PEO₁₁₄-p(HPMA₆₀-IbuPMA₂₀)

The targeted number average degree of polymerisation (DP_(n)) wasHPMA₆₀-IbuPMA₂₀. PEO₁₁₄-Br macroinitiator (0.59 g, 0.12 mmol 1 eq.) andHPMA (1 g, 6.94 mmol 60 eq.) and IbuPMA (0.77 g 2.3 mmol, 20 eq.) wereadded to a round-bottomed flask equipped with a nitrogen inlet/outletand a stirrer bar. Methanol was added (37 w/w %, based on HPMA+IbuPMA)and the solution was stirred vigorously under nitrogen for 10-15minutes. The copper catalyst Cu(I)Cl (0.0114 g, 0.12 mmol 1 eq.) and bpy(0.036 g, 0.23 mmol 2 eq.) were added to the flask and the temperaturewas fixed at 30° C. The reaction was monitored by ¹H NMR spectroscopyand terminated with methanol when the monomers had reached 99%conversion. The polymer was purified using a neutral alumina columnflushed with THF to remove excess copper catalyst. Excess THF wasremoved under vacuum to concentrate the sample before precipitation intocold petroleum ether 30/40. The resulting polymer was confirmed by ¹HNMR in MeOD, triple detection GPC with an eluent of THF.

Post Modification of PEO₁₁₄-HPMA₁₂₀—Targeted 40 HPMA Monomer Units

PEO₁₁₄-HPMA₁₂₀ (1 g, 0.033 mmol, 1 eq.), Ibuprofen (0.27 g, 1.33 mmol,40 eq.), DCC (0.274 g, 1.33 mmol, 40 eq.) and DMAP (1×10⁻³ g) weredissolved in THF (12 mL) and left over 24 hours. The DCU was filteredoff and washed with THF and concentrated in vacuo. The excess DMAP andDCU was dissolved in DCM and washed with 1M sodium bisulfate and driedwith MgSO4 and dried under vacuum.

ATRP Polymerisation—Branched IBU Modified Copolymerp(HPMA₆₀-IBU₂₀-EGDMA_(0.9))

The targeted degree of polymerisation (DP_(n)) was 50 repeat units.During a typical ATRP synthesis, EBIB initiator (0.024 g, 0.12 mmol 1eq.) and HPMA (1.04 g, 7.23 mmol 60 eq.) and IbuPMA (0.8 g, 2.41 mmol,20 eq.) were added to a round-bottomed flask equipped with a nitrogeninlet/outlet and a stirrer bar. Methanol was added (50 wt/wt %, based onHPMA) and the solution was stirred vigorously under nitrogen for 10-15minutes. The branching agent EGDMA (0.021 g, 0.11 mmol 0.9 eq. to EBIBinitiator), copper catalyst Cu(I)Cl (0.012 g, 0.12 mmol 1 eq.) and bpy(0.038 g, 0.24 mmol 2 eq.) were added to the flask and the temperaturewas fixed at 30° C. The reaction was monitored by ¹H NMR spectroscopyand terminated with methanol when the HPMA monomer had reached >99%conversion. The polymer was purified using a neutral alumina columnflushed with THF. Excess THF was removed under vacuum to concentrate thesample before precipitation. The polymer was precipitated from MeOH intocold petroleum ether 30/40. The resulting polymer was confirmed by ¹HNMR in MeOD, triple detection GPC with an eluent of THF.

Nanoparticle Preparation

IBU Modified Diblock—p(EO)₁₁₄-p(HPMA₆₀-HPMA:IBU₂₀):p(HPMA₅₀-EGDMA_(0.9))

During a typical nanoparticle preparation, 25 mg ofPEO₁₁₄-p(HPMA₆₀-HPMA:IBU₂₀) and 25 mg of p(HPMA₅₀-EGDMA_(0.9)) wereadded to 10 mL of MeOH during 6-8 hours to ensure completesolubilisation over 6-8 hours. 1 mL of the 5 mg/mL solution of polymerswas added to 5 mL of stirring distilled water (500 rpm) and left for 24hours for complete evaporation.

IBU Modified Branched Polymer Core p(HPMA₆₀-IBU₂₀-EGDMA_(0.9))

During a typical nanoparticle preparation, 25 mg of PEO₁₁₄-p(HPMA₁₂₀)and 25 mg of p(HPMA₆₀-IbuPMA₂₀-EGDMA_(0.9)) were added to 10 mL of MeOHduring 6-8 hours to ensure complete solubilisation over 6-8 hours. 1 mLof the 5 mg/mL solution of polymers was added to 5 mL of stirringdistilled water (500 rpm) and left for 24 hours for completeevaporation.

The same procedure can be repeated for other variations of thenanoparticles using the experimental above. For free IBU addition, thiscan simply be added into the methanol before solubilisation at thedesired ratio to allow mixtures of encapsulated IBU and IBU-prodrugswithin the particles.

Further Example Showing that a Different Type of Block Copolymer—aTri-Block A-B-A (Hydrophobic-Hydrophilic-Hydrophobic) Polymer—can bePrepared, and Used in Combination with a Branched Polymer and DrugEncapsulation

To illustrate a variation of the chemistry which may be used within thescope of the present invention, a bifunctional initiator was preparedand used as the “B” block in the preparation of an “A-B-A” tri-blockcopolymer. This tri-block copolymer was then used in combination with abranched vinyl polymer and drug molecules to form suitableco-nanoprecipitated particles.

Synthesis of PEG Bifunctional Macroinitiator

During a typical synthesis, OH-PEG₁₀₄-OH (M_(n) ˜4600 g, 1 equiv. 20 g,4.3 mmol), TEA (3 equiv. 0.013 mol, 1.83 mL) and DMAP (0.1 equiv, 4.0mmol, 0.053 g) were added to toluene (80 mL) in a two necked roundbottom flask fitted with a nitrogen inlet/outlet and stirrer bar.α-Bromo isobutyryl bromide (3 equiv. 13 mmol, 1.61 mL) was diluted intoluene (20 mL) and added drop-wise over 15 minutes via a droppingfunnel and left stirring over 24 hours. The reaction mixture was thenfiltered and the excess solvent removed in vacuo. The product wasdissolved in a minimal amount of acetone, and precipitated into coldpetroleum ether 40-60° C. (1:10 product:solvent) and left to dry undervacuum at 40° C. for 48 hours.

Synthesis of p(HPMA_(x)-b-PEG₁₀₄-b-HPMA_(x)) Block Polymer

During a typical synthesis, Br-PEG₁₀₄-Br (1 equiv. 2.17 g, 0.43 mmol),HPMA (80 equiv. 5.0 g, 34.6 mmol), anisole (0.1 ml) and anhydrousmethanol (50 wt %) were added to a round bottomed flask fitted with annitrogen inlet/outlet and stirrer bar. To this solution, anhydrousmethanol (50 wt %, based on HPMA), CuCl (1 equiv. 0.042 g, 0.43 mmol)and bpy (2 equiv. 0.135 g, 0.867 mmol) were added and the reactionmixture was degassed under nitrogen for 15-20 minutes. The reaction wasleft stirring for 24 hrs and high conversion was confirmed ¹H NMRspectra. The reaction was terminated by addition of methanol (˜50 ml)and the remaining solution was then passed through a neutral aluminacolumn to remove the catalytic system. The product was purified byconcentrating the solution in vacuo and precipitation of the polymerinto cold hexane (1:10 product: solvent). The resulting block copolymerwas analysed by ¹H NMR and GPC.

Branched p(DEAEMA₅₀) polymer was synthesized as previously described.Encapsulation of Drug Molecules into Co-Nanoprecipitated ParticlesConsisting of an A-B-A Triblock and a Branched Polymer Core

A stock solution of solubilised drug molecules (Ritonavir and Lopinavir)was added to a sample vial (up to 5 wt % of each) and the acetone wasleft to evaporate. To this vial, the branched polymer, A-B-A triblockpolymer (varying ratios of each) and acetone were added to make up asolution of 5 mg/mL. To ensure complete solubilisation this solution wasleft rolling over 24 hours. During a typical co-nanoprecipitation, 1 mLof the polymer/drug solution was rapidly added to 5 mL of stirringwater. The solution was left stirring overnight to ensure completesolvent removal and subsequent nanoparticles dispersions were measuredby dynamic light scattering.

Stable nanoparticles resulted, whether encapsulating Ritonavir alone,Lopinavir alone, or a 1:1 by weight mixture of both.

Particles Containing Branched Polymers and Linear Dendritic Hybrids

A second group of particles in accordance with the present invention arethose which contain branched polymers and linear dendritic hybrids.

Examples of Polymers Prepared by ATRP and of Combinations of SuchPolymers in Particles

Some non-limiting examples of components used in branched vinyl polymersand block copolymers have been described and defined above.

Linear dendritic hybrids may comprise monomers as referred to above, andadditionally, of course, comprise dendrons. In that context, somenon-limiting examples of components used in polymers of the presentinvention include the following.

-   -   G2-M: a generation 2 dendron initiator having the structure:

-   -   G2-Bz: a generation 2 dendron initiator with        benzyl-functionalised ends having the structure:

-   -   G1-M: a generation 1 dendron initiator having the structure:

-   -   G0-M: a generation 0 initiator having the structure:

Schematic representations of co-nanoprecipitates of branched polymersand linear dendritic hybrids are shown in FIG. 13. The dendrons areshown as wedges. In some examples (top right) the dendrons arehypothesised to be at least partially within the particles whereas inother examples (bottom right) the dendrons are hypothesised to be mainlyexposed at the surface. This can be controlled by varying pH, forexample when using pH responsive materials such as DEAEMA.

Some examples of combinations of polymers, and the properties of theresultant particles under different pH conditions, are shown in FIGS. 14to 18. FIGS. 14 to 18 use the abbreviation CNP which denotesco-nanoprecipitates. An SEM image is shown in FIG. 19 for the pH 7.8particles containing EBIB tBuMA-EGDMA and G2-M DEAEMA (FIG. 14).

^(a) All diameters are given as z-average values as measured by dynamiclight scattering.

^(b) All zeta potentials are given as surface charge values as measuredby dynamic light scattering

Further Examples of Combinations of Branched Vinyl Polymers and LinearDendritic Hybrids

Further examples of combinations of branched vinyl polymers and lineardendritic hybrids, prepared by ATRP, include those listed in thefollowing table. This exemplifies a range of chemistries (includinghydrophilic, hydrophobic, and pH responsive) and different copolymerapproaches (including block copolymers and statistical copolymers). Itwill also be seen that the block copolymer approach may be combined withthe linear dendritic hybrid approach, i.e. a linear dendritic hybrid maycomprise a dendron connected to one block which is then connected toanother block. Furthermore, in place of G2-M or G2-Bz, G1-M, G0-M orEBIB may be used instead, i.e. the linear chains may be initiated bysmaller dendrons or by initiators which are not dendrons (in which casethe materials co-precipitated with the branched vinyl polymer are notlinear dendritic hybrids but merely linear polymers, of which thepresent invention is concerned with a subset, namely block copolymers).

branched polymer linear dendritic hybrid EBIB-HPMA₅₀-EGDMA_(0.95)G2-M-HPMA₅₀ as above G2-M-DEAEMA₅₀ EBIB-tBuMA₅₀-EGDMA_(0.95)G2-M-DEAEMA₅₀ as above G2-M-HPMA₅₀ as above G2-M-PtBuMA₅₀ as above Blockcopolymers: G2-M-PDEAEMA₂₅-tBuMA₂₅ or G2-M-PDEAEMA₁₇-tBuMA₃₃ orG2-M-PDEAEMA₃₃-tBuMA₁₇ as above as above, except that the copolymers arerandom copolymers rather than block copolymers as above G2-Bz-DEAEMA₅₀EBIB-DEAEMA₅₀-EGDMA_(0.95) G2-M-DEAEMA₅₀ and EBIB-DEAEMA₅₀-BDME_(2.0) asabove EBIB-DEAEMA₅₀-EGDMA_(0.95) G2-M-HPMA₅₀EBIB-HPMA_(x)-DEAEMA_(y)-EGDMA_(0.9) G2-M-HPMA₅₀ (copolymer wherein x, y= 25, 25 or 17, 33 or 33, 17) as above G2-M-DEAEMA₅₀ as aboveG2-M-HPMA_(x)-DEAEMA_(y) (random copolymer wherein x, y = 17, 33 or 25,25 or 33, 17) as above (as above, except that the copolymer is a diblockcopolymer rather than a random copolymer) EBIB-HPMA₅₀-EGDMA_(0.95)G2-M-HPMA_(x)-DEAEMA_(y) (random copolymer wherein x, y = 17, 33 or 25,25 or 33, 17) as above (as above, except that the copolymer is a diblockcopolymer rather than a random copolymer) EBIB-DEAEMA₅₀-EGDMA_(0.95)G2-M-HPMA_(x)-DEAEMA_(y) (random copolymer wherein x, y = 17, 33 or 25,25 or 33, 17) as above (as above, except that the copolymer is a diblockcopolymer rather than a random copolymer)

The materials in the above table may for example be co-nanoprecipitatedin a ratio of 90:10 branched:linear. One suitable method involves:dropping 0.2 ml (5 mg ml⁻¹ in acetone, THF or IPA) of linear polymer and1.8 ml (5 mg ml⁻¹ in acetone, THF or IPA) of branched polymer into 10 mlwater; and allowing the organic solvent to evaporate overnight to form a1 mg ml⁻¹ nanoparticle dispersion in water.

A range of pH conditions may be used and optionally conditions andmonomers may be chosen to provide particular structures. Merely by wayof non-limiting example, at low pH protonation of amine moieties (e.g.in DEAEMA or in dendrons) means that amine moieties are more likely tobe exposed towards the outside of the particles

Examples of Polymers Prepared by Ring Opening Polymerization and of SuchPolymers in Particles

Another method of polymerization which can be used in the presentinvention is ring opening polymerisation (ROP). For example, lactonemonomers may be ring opened by reaction with alcohols under suitableconditions as known in the art.

For example, the polymerization of caprolactone monomer may be initiatedby benzyl alcohol to produce benzyl-polycaprolactone (abbreviated hereinas Bz-PCL).

The ring opening of single lactone rings such as polycaprolactoneresults in linear polymers.

Branched polymers may be obtained by using branchers, e.g. monomerswhich have two lactone rings connected together, e.g. BOD(4,4′-bioxepanyl-7,7′-dione). The following scheme shows a method ofpreparing BOD:

A branched polyester may be prepared by copolymerizing a monofunctionallactone (e.g. caprolactone) and a difunctional lactone (e.g. BOD) usingan initiator (e.g. Bz-OH):

Such branched polycaprolactones may be used instead of the branchedvinyl polymers, in combination with linear polymers (i.e. blockcopolymers or linear dendritic hybrids).

Examples of combinations of branched polycaprolactone (prepared by ROP)and linear dendritic hybrids (prepared by ATRP), which we haveco-nanoprecipitated, include those listed in the following table. Asbefore, in place of G2 it is possible to instead use G1, G0 or EBIB.

branched polycaprolactone linear dendritic hybrid Bz-PCL₅₀-BOD_(0.6)G2-M-DEAEMA₅₀ as above G2-M-HPMA₅₀ as above block copolymers:G2-M-DEAEMA₂₅-tBuMA₂₅ or G2-M-DEAEMA₁₇-tBuMA₃₃ or G2-M-DEAEMA₃₃-tBuMA₁₇as above as above except random copolymers instead of block copolymers

ROP may also be used to prepare the linear dendritic hybrid (or otherlinear polymer component), and ROP and ATRP may be combined.

Polyesters may also be used in the linear dendritic hybrid, andnon-limiting examples of suitable initiators for ROP in that contextinclude the following.

-   -   G2-pOH: a generation 2 dendron bearing a hydroxyl group to        initiate ROP, of the following structure:

-   -   G1-pOH: a generation 1 dendron bearing a hydroxyl group to        initiate ROP, of the following structure:

-   -   G0-pOH: a generation 0 initiator of the following structure:

Examples of linear dendritic hybrids which may be combined with branchedpolymers, e.g. Bz-PCL₅₀-BOD_(0.6), include:

-   -   G1-p-PCL₅₀-DEAEMA₂₀    -   G2-p-PCL₅₀    -   G2-p-PCL₃₀    -   G2-p-PCL₂₀

Example Experimental Procedures for ATRP and ROP Polymerisations and forthe Preparation of Materials Used in these Polymerisations 1.Polymerisation by ATRP 1.1 ATRP Dendron Initiator Synthesis 1.1.1Synthesis of G1-M-OH

2-(Dimethylamino)ethyl acrylate (6.0 g, 42 mmol, 6 eq.) was added to a50 mL round 2 necked round-bottomed flask containing isopropanol (IPA)(12 mL). The flask was deoxygenated under a positive N₂ purge for 10minutes. 1-amino-2-propanol (0.5246 g, 7.0 mmol, 1 eq.) dissolved in IPA(12 mL) was added drop wise while the solution was stirring in an icebath under a positive flow of N₂. The final mixture was stirred for afurther 10 minutes at 0° C. before being allowed to warm to roomtemperature and left stirring for 48 hrs. The solvent was removed andthe product left to dry in vacuo overnight. Found C, 57.45; H, 9.77; N,11.12%. C₁₇H₃₅N₃O₅ requires, C, 56.43; H, 9.68; N, 11.62%. ¹H NMR (400MHz, CDCl₃) δ 1.08 (d, 3H), 2.18-2.62 (m, 22H), 2.69 (m, 2H), 2.89 (m,2H), 3.77 (m, 1H), 4.16 (m, 4H). ¹³C NMR (100 MHz, CDCl3) δ 19.8, 32.6,45.6, 49.7, 57.8, 62.0, 63.7, 76.9, 128.4, 130.9, 172.5. m/z (ES MS)362.3 [M+H]+, 384.3 [M+Na]+.

1.1.2 Synthesis of G2-M-OH

2-(Dimethylamino)ethyl acrylate (6.0 g, 42 mmol, 6 eq.) was added to a50 mL round 2 necked round-bottomed flask containing IPA (12 mL). Theflask was deoxygenated under a positive N₂ purge for 10 minutes.Bis(3-aminopropyl)amino)propan-2-ol (1.3221 g, 6.984 mmol, 1 eq.)dissolved in IPA (12 mL) was added drop wise while the solution wasstirring in an ice bath under a positive flow of N₂. The final mixturewas stirred for a further 10 minutes at 0° C., allowed to warm to roomtemperature and left stirring for 48 hrs. The solvent was removed andthe product left to dry in vacuo overnight. Found C, 58.32; H, 9.92; N,12.87%. C₃₇H₇₅N₇O₉ requires, C, 58.27; H, 9.84; N, 12.86%. ¹H NMR (400MHz, CDCl₃) δ 1.13 (d, 3H), 1.67 (m, 4H), 2.26-2.65 (m, 50H), 2.77 (m,8H), 3.87 (m, 1H), 4.17 (m, 8H). m/z (ES MS) 762.6 [M+H]+, 784.6[M+Na]+.

1.1.3 Synthesis of G0-M

1-dimethylamino-2-propanol (1.1207 g, 10.86 mmol, 1 eq.),triethanolamine (TEA) (1.5390 g, 15.2 mmol, 1.4 eq.) and dimethyl aminopyridine (DMAP) (132.7 mg, 1.086 mmol, 0.1 eq.) were added to a 250 mL 2necked round-bottomed flask containing dichloromethane (DCM) (160 mL).The flask was deoxygenated under a positive N₂ purge for 10 minutes.α-bromoisobutyryl bromide (2.622 g, 1.4 mL, 11.4 mmol, 1.05 eq.) wasadded drop wise while the solution was stirring in an ice bath under apositive flow of N₂. The reaction mixture was allowed to warm to roomtemperature and left stirring overnight. The organic phase was washedwith saturated sodium hydrogen carbonate (NaHCO₃) solution (3×30 mL).The solution was dried with anhydrous Na₂SO₄. Found C, 42.87; H, 7.20;N, 5.55%. C₉H₁₈NO₂Br requires, C, 42.86; H, 7.14; N, 5.55%. ¹H NMR (400MHz, CDCl₃) δ 1.27 (d, 3H), 1.89 (s, 6H), 2.17-2.55 (m, 8H), 5.07 (m,1H). ¹³C NMR (100 MHz, CDCl₃) δ 17.6, 30.9, 46.1, 56.1, 63.5, 70.6,76.9, 170.8. m/z (ES MS) 252 [M+H]+.

1.1.4 Synthesis of G1-M

G1-OH (1.1207 g, 10.86 mmol, 1 eq.), TEA (1.5390 g, 15.2 mmol, 1.4 eq.)and DMAP (132.7 mg, 1.086 mmol, 0.1 eq.) were added to a 250 mL 2 neckedround-bottomed flask containing DCM (160 mL). The flask was deoxygenatedunder a positive N₂ purge for 10 minutes. α-bromoisobutyryl bromide(2.622 g, 1.4 mL, 11.4 mmol, 1.05 eq.) was added drop wise while thesolution was stirring in an ice bath under a positive flow of N₂. Thereaction mixture was allowed to warm to room temperature and leftstirring overnight. The organic phase was washed with saturated sodiumhydrogen carbonate (NaHCO₃) solution (3×160 mL). The solution was driedwith anhydrous Na₂SO₄ and the product left to dry in vacuo overnight.Found C, 49.41; H, 7.90; N, 8.23%. C₂₁H₄₀N₃O₆Br requires, C, 49.41; H,7.84; N, 8.24%. ¹H NMR (400 MHz, CDCl₃) δ 1.22 (d, 3H), 1.89 (s, 6H),2.24-2.69 (m, 22H), 2.83 (m, 4H), 4.20 (m, 4H), 5.0 (m, 1H). ¹³C NMR(100 MHz, CDCl3) δ 18.6, 30.9, 32.8, 50.0, 56.4, 58.8, 60.3, 69.6, 77.2,125.7, 144.3, 172.3. m/z (ES MS) 510.2 [M+H]+, 534.2 [M+Na]+.

1.1.5 Synthesis of G2-M

G2-OH (5.1431 g, 6.749 mmol, 1 eq.), TEA (0.9561 g, 9.449 mmol, 1.4 eq.)and DMAP (82.5 mg, 0.6749 mmol, 0.1 eq.) were added to a 250 mL 2 neckedround-bottomed flask containing DCM (160 mL). The flask was deoxygenatedunder a positive N₂ purge for 10 minutes. α-bromoisobutyryl bromide(1.629 g, 0.88 mL, 7.087 mmol, 1.05 eq.) was added drop wise while thesolution was stirring in an ice bath under a positive flow of N₂. Thereaction mixture was allowed to warm to room temperature and leftstirring overnight. The organic phase was washed with saturated sodiumhydrogen carbonate (NaHCO₃) solution (3×160 mL). The solution was driedwith anhydrous Na₂SO₄ and the product left to dry in vacuo overnight.Found C, 54.05; H, 8.85; N, 10.76%. C₄₁H₈₀N₇O₁₀Br requires, C, 54.01; H,8.78; N, 10.76%. ¹H NMR (400 MHz, CDCl₃) δ 1.26 (d, 3H), 1.56 (m, 4H),1.91 (s, 6H), 2.22-2.67 (m, 50H), 2.76 (m, 8H), 4.19 (m, 8H), 5.04 (m,1H). ¹³C NMR (100 MHz, CDCl3) δ 24.5, 25.5, 28.4, 45.6, 62.2, 64.2,77.2, 173.5. m/z (ES MS) 912.5 [M+H]+, 934.5 [M+Na]+, 950.5 [M+K]+.

1.1.6 Synthesis of G2-Bz-OH

Benzyl acrylate (6.7966 g, 42 mmol, 6 eq.) was added to a 50 mL round 2necked round-bottomed flask containing IPA (12 mL). The flask wasdeoxygenated under a positive N₂ purge for 10 minutes.Bis(3-aminopropyl)amino)propan-2-ol (1.3221 g, 6.984 mmol, 1 eq.)dissolved in IPA (12 mL) was added drop wise while the solution wasstirring in an ice bath under a positive flow of N₂. The final mixturewas stirred for a further 10 minutes at 0° C., allowed to warm to roomtemperature and left stirring for 48 hrs. The solvent was removed andthe product left to dry in vacuo overnight.

1.1.7 Synthesis of G2-Bz

G2-Bz (1.664 g, 1.86 mmol, 1 eq.), TEA (0.2639 g, 2.6 mmol, 1.4 eq.) andDMAP (22.8 mg, 0.1866 mmol, 0.1 eq.) were added to a 250 mL 2 neckedround-bottomed flask containing DCM (110 mL). The flask was deoxygenatedunder a positive N₂ purge for 10 minutes. α-bromoisobutyryl bromide(0.5354 g, 0.29 mL, 2.329 mmol, 1.25 eq.) was added drop wise while thesolution was stirring in an ice bath under a positive flow of N₂. Thereaction mixture was allowed to warm to room temperature and leftstirring overnight. The organic phase was washed with saturated sodiumhydrogen carbonate (NaHCO₃) solution (3×110 mL). The solution was driedwith anhydrous Na₂SO₄ and the product left to dry in vacuo overnight.

Found C, 63.40; H, 6.96; N, 4.18%. C₅₃H₆₈N₃O₁₀Br requires, C, 64.44; H,6.89; N, 4.26%. ¹H NMR (400 MHz, CDCl₃) δ 1.22 (d, 3H), 1.54 (m, 4H),1.90 (s, 6H), 2.24-2.65 (m, 18H), 2.77 (m, 8H), 5.00 (m, 1H), 5.09 (s,8H), 7.33 (m, 20H). ¹³C NMR (100 MHz, CDCl3) δ 18.1, 30.6, 32.3, 44.1,48.8, 51.7, 52.7, 66.7, 76.7, 128.6, 135.8, 144.1, 172.3. m/z (ES MS)988.4 [M+H]+.

1.2 pH Responsive Brancher Synthesis 1.2.1 Synthesis of 1,4-Butanedioldi(methacryoyloxy)-ethyl ether (BDME)

1,4-butanediol divinyl ether (BDVE) (5.6 ml, 35.21 mmol) was added to atwo-necked 250 ml round bottomed flask equipped with a condenser, amagnetic stirrer and a positive flow of nitrogen. A small amount ofradical inhibitor 4-tert-butylcatechol (end of a spatula) was added andthe mixture deoxygenated using a nitrogen purge for 15 minutes. Oncedissolved, the temperature was raised to 70° C. Methacrylic acid (MAA)(14.9 ml, 175.8 mmol) was added dropwise over 10 minutes through asepta. The reaction was allowed to proceed at 70° C. for a further 6hours with stirring. After this time, the reaction was stopped bycooling and exposing to the air. The crude product was dissolved inchloroform (100 ml) and washed with basic H₂O (˜pH12, 3×100 ml). Thecombined washings were collected and dried over NaSO₄ and the solventremoved by rotary evaporation.

(Found: C 61.45; H 8.28%. C₁₆H₂₆O₆ requires C 61.15; H 8.28%); ¹H NMR(400 MHz; CDCl₃; Me₄Si) δ 1.44 (6H, d, CH₃CH), 1.65 (4H, m, CH₂CH₂CH₂),1.95 (6H, s, CH₃C═CH2), 3.50-3.69 (4H, m, OCH₂CH₂), 5.60 and 6.15 (4H,2s, CH₂═CCH₃) and 5.95-5.99 (2H, q, CHCH₃). ¹³C NMR (400 MHz; CDCl₃;Me₄Si) δ 18.27 (s), 20.83 (s), 26.29 (s) 68.85 (s), 96.93 (s), 125.90(s), 136.37 (s) and 167.01 (s). m/z (EI) 314.2 (M⁺-C₁₆H₂₆O₆ requires314).

1.3 Polymerisation of HPMA 1.3.1 Polymerisation of HPMA₅₀

In a typical synthesis, targeting a number average degree ofpolymerisation (DP_(n))=50 monomer units P(HPMA)₅₀;n_(HPMA)/n_(Initiator): 50), bpy (173.3 mg, 1.1096 mmol, 2 eq.), HPMA (4g, 27.7 mmol, 50 eq.) and methanol (MeOH) (56% v/v based on HPMA) wereplaced into a 25 mL round-bottomed flask. The solution was stirred anddeoxygenated using a nitrogen (N₂) purge for 15 minutes. Cu(I)Cl (54.9mg, 0.5548 mmol, 1 eq.) was added to the flask and left to purge for afurther 5 minutes. G2-M (0.5054 g, 0.5548 mmol, 1 eq.) was added to theflask under a positive flow of N₂, and the solution was left topolymerise at 30° C. Reactions were terminated when >99% conversion wasreached, as judged by ¹H NMR (cf. FIG. 8), by exposure to oxygen andaddition of THF. The catalyst residues were removed by passing themixture over a basic alumina column. THF was removed under vacuum toconcentrate the sample before precipitation into hexane and drying inthe vacuum oven overnight.

1.3.2 Polymerisation of HPMA₅₀-EGDMA_(x)

In a typical synthesis, targeting a number average degree ofpolymerisation (DP_(n))=50 monomer units P(HPMA)₅₀;n_(HPMA)/n_(Initiator): 50), bpy (173.3 mg, 1.1096 mmol, 2 eq.), HPMA (4g, 27.7 mmol, 50 eq.), EGDMA (99.0 mg, 0.4993 mmol, 0.9 eq) and methanol(MeOH) (38.9% v/v based on HPMA) were placed into a 25 mL round-bottomedflask. The solution was stirred and deoxygenated using a nitrogen (N₂)purge for 15 minutes. Cu(I)Cl (54.9 mg, 0.5548 mmol, 1 eq.) was added tothe flask and left to purge for a further 5 minutes. EBIB (0.1082 g,0.5548 mmol, 1 eq.) was added to the flask under a positive flow of N₂,and the solution was left to polymerise at 30° C. Reactions wereterminated when >99% conversion was reached, as judged by ¹H NMR, byexposure to oxygen and addition of THF. The catalyst residues wereremoved by passing the mixture over a basic alumina column. THF wasremoved under vacuum to concentrate the sample before precipitation intohexane.

1.4 Polymerisation of DEAEMA 1.4.1 Polymerisation of DEAEMA₅₀

In a typical synthesis, targeting a DP_(n)=50 monomer units PDEAEMA₅₀;n_(DEAEMA)/n_(Initiator): 50), bpy (134.9 mg, 0.8637 mmol, 2 eq.),DEAEMA (4 g, 21.59 mmol, 50 eq.) and IPA (56% v/v based on DEAEMA) wereplaced into a 25 mL round-bottomed flask. The solution was stirred anddeoxygenated using a nitrogen (N₂) purge for 15 minutes. Cu(I)Cl (42.8mg, 0.4318 mmol, 1 eq.) was added to the flask and left to purge for afurther 5 minutes. G2-M (0.3934 g, 0.4318 mmol, 1 eq.) was added to theflask under a positive flow of N₂, and the solution was left topolymerise at 40° C. Reactions were terminated when >99% conversion wasreached, as judged by ¹H NMR, by exposure to oxygen and addition ofacetone. The catalyst residues were removed by passing the mixture overa basic alumina column. Acetone was removed under vacuum to concentratethe sample before precipitation into cold petroleum ether (40° C.-60°C.) and drying in the vacuum oven overnight.

1.4.2 Polymerisation of DEAEMA₅₀-EGDMA_(x)

In a typical synthesis, targeting a DP_(n)=50 monomer units PDEAEMA₅₀;n_(DEAEMA)/n_(Initiator): 50), bpy (134.9 mg, 0.8637 mmol, 2 eq.),DEAEMA (4 g, 21.59 mmol, 50 eq.), EGDMA (77.0 mg, 0.3886 mmol, 0.9 eq)and IPA (38.9% v/v based on DEAEMA) were placed into a 25 mLround-bottomed flask. The solution was stirred and deoxygenated using aN₂ purge for 15 minutes. Cu(I)Cl (42.8 mg, 0.4318 mmol, 1 eq.) was addedto the flask and left to purge for a further 5 minutes. EBIB (84.2 mg,0.4318 mmol, 1 eq.) was added to the flask under a positive flow of N₂,and the solution was left to polymerise at 40° C. Reactions wereterminated when >99% conversion was reached, as judged by ¹H NMR, byexposure to oxygen and addition of acetone. The catalyst residues wereremoved by passing the mixture over a basic alumina column. Acetone wasremoved under vacuum to concentrate the sample before precipitation intocold petroleum ether (40° C.-60° C.) and drying in the vacuum ovenovernight. The polymerisation conditions and procedure is identical tothose described for linear polymers above.

1.4.3 Polymerisation of DEAEMA₅₀-BDME_(2.0)

In a typical synthesis, targeting a DP_(n))=50 monomer units PDEAEMA₅₀;n_(DEAEMA)/n_(Initiator): 50), bpy (134.9 mg, 0.8637 mmol, 2 eq.),DEAEMA (4 g, 21.59 mmol, 50 eq.), BDME (291.2 mg, 0.8637 mmol, 0.9 eq)and IPA (38.9% v/v based on DEAEMA) were placed into a 25 mLround-bottomed flask. The solution was stirred and deoxygenated using aN₂ purge for 15 minutes. Cu_((I))Cl (42.8 mg, 0.4318 mmol, 1 eq.) wasadded to the flask and left to purge for a further 5 minutes. EBIB (84.2mg, 0.4318 mmol, 1 eq.) was added to the flask under a positive flow ofN₂, and the solution was left to polymerise at 40° C. Reactions wereterminated when >99% conversion was reached, as judged by ¹H NMR, byexposure to oxygen and addition of acetone. The catalyst residues wereremoved by passing the mixture over a basic alumina column. Acetone wasremoved under vacuum to concentrate the sample before precipitation intocold petroleum ether (40° C.-60° C.) and drying in the vacuum ovenovernight. The polymerisation conditions and procedure is identical tothose described for linear polymers above.

1.5 Polymerisation of tBuMA 1.5.1 Polymerisation of tBuMA₅₀

In a typical synthesis, targeting a number average degree ofpolymerisation (DP_(n))=50 monomer units (tBuMA₅₀);n_(tBumA)/n_(Initiator): 50), bpy (175.7 mg, 1.125 mmol, 2 eq.), tBuMA(4 g, 28.129 mmol, 50 eq.) and aqueous isopropanol (IPA/H₂O) (92.5/7.5%)(50.4% v/v based on tBuMA) were placed into a 50 mL round-bottomedflask. The solution was stirred and deoxygenated using a nitrogen (N₂)purge for 15 minutes. Cu(I)Cl (55.7 mg, 0.5626 mmol, 1 eq.) was added tothe flask and left to purge for a further 5 minutes. G2-M (0.3934 g,0.5626 mmol, 1 eq.) was added to the flask under a positive flow of N₂,and the solution was left to polymerise at 20° C. and samples were takenperiodically from the reaction mixture for ¹H NMR analysis. Reactionswere terminated when >99% conversion was reached, as judged by ¹H NMR,by exposure to oxygen and addition of THF. The catalyst residues wereremoved by passing the mixture over a basic alumina column. THF wasremoved under vacuum to concentrate the sample before precipitation intohexane and drying in the vacuum oven overnight.

1.5.2 Polymerisation of tBuMA₅₀-EGDMA_(0.95)

In a typical synthesis, targeting a number average degree ofpolymerisation (DP_(n))=50 monomer units (tBuMA₅₀);n_(tBumA)/n_(Initiator): 50), bpy (175.7 mg, 1.125 mmol, 2 eq.), tBuMA(4 g, 28.13 mmol, 50 eq.), EGDMA (105.9 mg, 0.5345 mmol) and aqueousisopropanol (IPA/H₂O) (92.5/7.5%) (38.4% v/v based on tBuMA) were placedinto a 50 mL round-bottomed flask. The solution was stirred anddeoxygenated using a nitrogen (N₂) purge for 15 minutes. Cu(I)Cl (55.7mg, 0.5626 mmol, 1 eq.) was added to the flask and left to purge for afurther 5 minutes. EBIB (0.1097 g, 0.5626 mmol, 1 eq.) was added to theflask under a positive flow of N₂, and the solution was left topolymerise at 20° C. and samples were taken periodically from thereaction mixture for ¹H NMR analysis. Reactions were terminatedwhen >99% conversion was reached, as judged by ¹H NMR, by exposure tooxygen and addition of THF. The catalyst residues were removed bypassing the mixture over a basic alumina column. THF was removed undervacuum to concentrate the sample before precipitation into hexane anddrying in the vacuum oven overnight.

1.6 Polymerisation of DEAEMA-tBuMA 1.6.1 Polymerisation ofG2-DEAEMA_(x)-Stat-tBuMA_(y)

In a typical synthesis, targeting a DP_(n)=50 monomer unitsPDEAEMA₂₅-tBuMA₂₅; n_(DEAEMA)/n_(Initiator): 50), bpy (134.9 mg, 0.8637mmol, 2 eq.), DEAEMA (2 g, 10.80 mmol, 25 eq.), tBuMA (1.5352, 10.80mmol, 25 eq.) and IPA/H₂O (92.5/7.5%) (30.6% v/v based on DEAEMA/tBuMA)were placed into a 50 mL round-bottomed flask. The solution was stirredand deoxygenated using a nitrogen (N₂) purge for 15 minutes. Cu(I)Cl(42.8 mg, 0.4318 mmol, 1 eq.) was added to the flask and left to purgefor a further 5 minutes. G2-M (0.3934 g, 0.4318 mmol, 1 eq.) was addedto the flask under a positive flow of N₂, and the solution was left topolymerise at 40° C. Reactions were terminated when >99% conversion wasreached, as judged by ¹H NMR, by exposure to oxygen and addition ofacetone. The catalyst residues were removed by passing the mixture overa basic alumina column. Acetone was removed under vacuum to concentratethe sample before precipitation into cold petroleum ether (40° C.-60°C.) and drying in the vacuum oven overnight.

1.6.2 Polymerisation of EBIB-DEAEMA_(x)-Stat-tBuMA_(y)EGDMA_(0.9)

In a typical synthesis, targeting a DP_(n)=50 monomer unitsPDEAEMA₂₅-tBuMA₂₅; n_(DEAEMA)/n_(Initiator): 50), bpy (134.9 mg, 0.8637mmol, 2 eq.), DEAEMA (2 g, 10.80 mmol, 25 eq.), tBuMA (1.5352, 10.80mmol, 25 eq.), EGDMA (77 mg, 0.9 mmol, 0.9 eq.) and IPA/H₂O (92.5/7.5%)(30.6% v/v based on DEAEMA/tBuMA) were placed into a 50 mLround-bottomed flask. The solution was stirred and deoxygenated using anitrogen (N₂) purge for 15 minutes. Cu(I)Cl (42.8 mg, 0.4318 mmol, 1eq.) was added to the flask and left to purge for a further 5 minutes.EBIB (84.2 mg, 0.4318 mmol, 1 eq.) was added to the flask under apositive flow of N₂, and the solution was left to polymerise at 40° C.Reactions were terminated when >99% conversion was reached, as judged by¹H NMR, by exposure to oxygen and addition of acetone. The catalystresidues were removed by passing the mixture over a basic aluminacolumn. Acetone was removed under vacuum to concentrate the samplebefore precipitation into cold petroleum ether (40° C.-60° C.) anddrying in the vacuum oven overnight.

1.6.3 Polymerisation of G2-DEAEMA_(x)-Block-tBuMA_(y)

In a typical synthesis, targeting a DP_(n)=50 monomer unitsPDEAEMA₂₅-tBuMA₂₅; n_(DEAEMA)/n_(Initiator): 50), bpy (134.9 mg, 0.8637mmol, 2 eq.), DEAEMA (4 g, 21.59 mmol, 50 eq.) and IPA (38.9% v/v basedon DEAEMA) were placed into a 50 mL round-bottomed flask. The solutionwas stirred and deoxygenated using a nitrogen (N₂) purge for 15 minutes.Cu(I)Cl (42.8 mg, 0.4318 mmol, 1 eq.) was added to the flask and left topurge for a further 5 minutes. G2-M (0.3934 mg, 0.4318 mmol, 1 eq.) wasadded to the flask under a positive flow of N₂, and the solution wasleft to polymerise at 40° C. The reaction was allowed to reach 80-90%conversion before tBuMA (1.5352, 10.80 mmol, 25 eq.), bpy (134.9 mg,0.8637 mmol, 2 eq.) and Cu(I)Cl (42.8 mg, 0.4318 mmol, 1 eq.) dissolvedin H₂O/IPA (92.5/7.5%) (27.3% v/v based on tBuMA) was added to thereaction mixture and left to polymerise overnight. Reactions wereterminated when >99% conversion was reached, as judged by ¹H NMR, byexposure to oxygen and addition of acetone. The catalyst residues wereremoved by passing the mixture over a basic alumina column. Acetone wasremoved under vacuum to concentrate the sample before precipitation intocold petroleum ether (40° C.-60° C.) and drying in the vacuum ovenovernight.

1.6.4 Polymerisation of EBIB-DEAEMA_(x)-Block-tBuMA_(y)EGDMA_(0.9)

In a typical synthesis, targeting a DP_(n)=50 monomer unitsPDEAEMA₂₅-tBuMA₂₅; n_(DEAEMA)/n_(Initiator): 50), bpy (134.9 mg, 0.8637mmol, 2 eq.), DEAEMA (4 g, 21.59 mmol, 50 eq.) and IPA (38.9% v/v basedon DEAEMA) were placed into a 50 mL round-bottomed flask. The solutionwas stirred and deoxygenated using a nitrogen (N₂) purge for 15 minutes.Cu(I)Cl (42.8 mg, 0.4318 mmol, 1 eq.) was added to the flask and left topurge for a further 5 minutes. EBIB (84.2 mg, 0.4318 mmol, 1 eq.) wasadded to the flask under a positive flow of N₂, and the solution wasleft to polymerise at 40° C. The reaction was allowed to reach 80-90%conversion before tBuMA (1.5352, 10.80 mmol, 25 eq.), bpy (134.9 mg,0.8637 mmol, 2 eq.), EGDMA (77 mg, 0.9 mmol, 0.9 eq.) and Cu(I)Cl (42.8mg, 0.4318 mmol, 1 eq.) dissolved in H₂O/IPA (92.5/7.5%) (27.3% v/vbased on tBuMA) was added to the reaction mixture and left to polymeriseovernight. Reactions were terminated when >99% conversion was reached,as judged by ¹H NMR, by exposure to oxygen and addition of acetone. Thecatalyst residues were removed by passing the mixture over a basicalumina column. Acetone was removed under vacuum to concentrate thesample before precipitation into cold petroleum ether (40° C.-60° C.)and drying in the vacuum oven overnight.

2. Polymerisation by ROP 2.1 ROP Dendron Initiator Synthesis 2.1.1Synthesis of G1-pOH

2-(Dimethylamino)ethyl acrylate (6.0 g, 42 mmol, 6 eq.) was added to a50 mL round 2 necked round-bottomed flask containing IPA (12 mL). Theflask was deoxygenated under a positive N₂ purge for 10 minutes.Ethanolamine (0.4266 g, 6.9843 mmol, 1 eq.) dissolved in IPA (12 mL) wasadded drop wise while the solution was stirring in an ice bath under apositive flow of N₂. The final mixture was stirred for a further 10minutes at 0° C. before being allowed to warm to room temperature andleft stirring for 48 hrs. The solvent was removed and the product leftto dry in vacuo overnight.

2.1.2 Synthesis of G2-pOH

CDI (39.137 g, 0.241 mol) was added to a 500 mL 2-neck RBF fitted with areflux condenser, magnetic stirrer and a dry N₂ inlet. Dry toluene (350mL) was added and the flask was purged with N₂ for 10 minutes. Thesolution was stirred at 60° C. and t-Butanol (35.7 g, 46 mL, 0.483 mol)was added via a warm syringe (Note: t-Butanol is a low melting solid,hence warm it in a water bath at 35° C. to allow it to melt to a liquidto easily get it out of the bottle by syringe). The mixture was leftstirring at 60° C. for 6 hours under a positive flow of nitrogen.Following this, BAPA (16.077 g, 17.14 mL, 0.121 mol) was added dropwisewhilst stirring and maintaining the temperature at 60° C. Upon addition,a white solid precipitate began to form in the flask (imidazole). Thereaction was left stirring for a further 18 hours at 60° C. under apositive flow of nitrogen, and then allowed to cool to room temperature.The pale yellow solution was filtered to remove any solid imidazole, andconcentrated in vacuo. The remaining oil was dissolved indichloromethane (250 mL) washed with distilled water (3×250 mL) andfinally a saturated brine solution (150 mL). The organic layer was driedwith anhydrous Na₂SO₄, filtered and concentrated in vacuo to giveG1-BAPA as a white solid powder. To remove any remaining residualsolvents, the compound was placed under high vacuum overnight. (38 g,95%) Found C, 57.84; H, 10.45; N, 12.91%. C₁₆H₃₃N₃O₄ requires, C, 57.98;H, 10.04; N, 12.68%. ¹H NMR (400 MHz, CDCl₃) 5.19 (s, br, NH—disappearson addition of D₂O), 3.21 (t, 4H), 2.65 (t, 4H), 1.65 (q, 4H), 1.44 (s,18H)¹³C NMR (100 MHz, CDCl₃) 156.48, 79.34, 47.77, 39.29, 30.11, 28.79.m/z (ES MS) 332.3 [M+H]⁺

A mixture of G1-BAPA (20 g, 0.06 mol) 16 mmol) in 1,4-dioxane (200 mL),bromoethanol (7.54 g, 0.6 mol), 30 mg of sodium iodide, and potassiumcarbonate (25.0 g, 1.8 mol) was refluxed overnight. After concentrationof the reaction mixture, it was extracted with ethyl acetate (200 mL),washed with water (100 mL), dried over sodium sulfate, and filtered, andthe solvent was removed under reduced pressure. Purification of thecrude product by flash chromatography (2:1, ethyl acetate-hexane)produced G1-OH.

In a 1 L RBF, G1-OH (33.70 g) was dissolved in ethyl acetate (330 mL)and concentrated HCl (35.03 g, 30 mL, d=1.18 36% active) was added veryslowly. CO₂ began to evolve. The reaction vessel was left open andstirring for 6 hours. The ethyl acetate was then removed in vacuo, and acrude ¹H NMR (D₂O) of the remaining oil taken. The crude ¹H NMR showedsigns of incomplete decarboxylation (see page 7 for spectra), so the oilwas re-dissolved in 250 mL ethyl acetate and heated to 55° C. for 5hours. After removal of ethyl acetate, the crude oil was dissolved in 4MNaOH (300 mL), and then reduced down by half (approx.) on the rotaryevaporator (60° C.). Following this, the oily mixture was extractedtwice with CHCl₃ (300 mL). The organic layers were then combined, driedwith anhydrous Na₂SO₄, filtered and concentrated in vacuo to givebis(3-aminopropyl)amino)propanol as a pale yellow oil.

2-(Dimethylamino)ethyl acrylate (6.0 g, 42 mmol, 6 eq.) was added to a50 mL round 2 necked round-bottomed flask containing IPA (12 mL). Theflask was deoxygenated under a positive N₂ purge for 10 minutes.Bis(3-aminopropyl)amino)propanol (1.2271 g, 6.984 mmol, 1 eq.) dissolvedin IPA (12 mL) was added drop wise while the solution was stirring in anice bath under a positive flow of N₂. The final mixture was stirred fora further 10 minutes at 0° C., allowed to warm to room temperature andleft stirring for 48 hrs. The solvent was removed and the product leftto dry in vacuo overnight.

2.2 Synthesis of Bifunctional Caprolactone 2.2.2 Synthesis of4,4′-bioxepanyl-7,7′-dione (BOD)

Urea hydrogen peroxide (CO(NH₂).H₂O₂) (20 g, 0.21 mol) was added to a500 mL round-bottom flask containing formic acid (100 mL). The solutionwas stirred for 2 h at room temperature. The flask was then immersed inan ice bath to control the exotherm resulting from the following stageof the procedure. Bicyclohexanone (10 g, 0.05 mol) was slowly added tothe solution over a period of 5-10 min. The reaction mixture was stirredfor 4 h whilst the ice bath was changed periodically. 200 mL of waterwas then added to the mixture followed by extraction with chloroform(200 mL×4). The organic fraction was collected and washed with asaturated aqueous sodium bicarbonate solution then dried overnight withNa₂SO₄. After removing the solvent under reduced pressure, a whitepowder was isolated and analysed by NMR and compared to the literaturereported values.

2.3 Polymerisation by ROP 2.3.1 Ring Opening Polymerisation ofε-Caprolactone

The typical protocol for the homopolymerisation of CL for a targetnumber average degree of polymerization DP=50 was as follows. A 50 mLsingle necked round bottomed flask was purged with nitrogen for 15minutes. SnOct₂ catalyst (0.0018 g, 0.0044 mmol) was added by syringeand needle and the flask purged further. Distilled CL (3.773 g, 3.5 mL,33.06 mmol) was introduced into a 50 mL flask and the flask purged for afurther 10 minutes. Anhydrous G1pOH (0.2316 g, 0.6666 mmol) was addedvia syringe. The flask was then immersed in a preheated oil bath at 110°C. and vigorously stirred for the required reaction time of ˜20 hours.The reaction was killed by submerging the reaction in an ice bath andthe polymer purified by dissolving in THF and precipitating into hexane.

2.3.2 Ring Opening Polymerisation of ε-Caprolactone and4,4′-Bioxepanyl-7,7′-Dione (PCL-BOD)

The typical protocol for the homopolymerisation of CL for a targetnumber average degree of polymerization DP=50 was as follows. A 50 mLsingle necked round bottomed flask containing BOD (0.9183 g 4.059 mmol)was purged with nitrogen for 15 minutes. SnOct₂ catalyst (0.0079 g,0.0195 mmol) was added by syringe and needle and the flask purgedfurther. Distilled CL (23.28 g, 21.6 mL, 204 mmol) was introduced into a50 mL flask and the flask purged for a further 10 minutes. AnhydrousBzOH (0.7315 g, 0.7 mL, 6.764 mmol) was added via syringe. The flask wasthen immersed in a preheated oil bath at 110° C. and vigorously stirredfor the required reaction time of ˜20 hours. The reaction was killed bysubmerging the reaction in an ice bath and the polymer purified bydissolving in THF and precipitating into hexane.

In Vitro Cell Viability Experiments

The effect of nanoparticles carrying SN38, in accordance with thepresent invention, on the viability of murine CT-26 cells, wasinvestigated. It was found that, whilst SN38 is responsible for areduction in cell viability, the nanoparticle carrier itself does notaffect cell viability.

The following polymer compositions (mixtures of branched polymers andblock copolymers) were used:

JF1: p(HPMA₅₀-co-EGDMA_(0.9)):p(PEG₁₁₄-b-HPMA₁₂₀) 50:50 wt %JF2: p(nBuMA₅₀-co-EGDMA_(0.8)):p(PEG₁₁₄-b-HPMA₁₂₀) 50:50 wt %JF3: p(tBuMA₂₅-co-HPMA₂₅-co-EGDMA_(0.9)):p(PEG₁₁₄-b-HPMA₁₂₀) 50:50 wt %The following protocol was followed.

Nanoparticle Samples—5 wt % Encapsulation of SN-38

-   -   1. Add 30 uL of sample (JF1-3) to 970 uL media (final        concentration is 4 uM)    -   2. Add 15 uL of sample (JF1-3) to 970 uL media and 15 uL water        (final concentration is 2 uM)    -   3. Add 7.5 uL of sample (JF1-3) to 970 uL media and 22.5 uL        water (final concentration is 1 uM)

For SN38 in DMSO

-   -   1. Make 2.5 mM stock SN38 solution (2 mg in 2000 uL DMSO)    -   2. Add 1.6 uL of stock to 968.4 uL media and then add 30 uL        sterile water (final concentration is 4 uM)    -   3. Add 2 uL of stock to 2 ul of DMSO (serial dilution of 1:2),        add 1.6 uL of this to 968.4 uL media and 30 uL sterile water        (final concentration is 2 uM)    -   4. Add 2 uL of previous intermediate stock to 2 uL DMSO (serial        dilution of 1:2), add 1.6 uL of this to 968.4 uL media and 30 uL        sterile water (final concentration is 1 uM)

For Controls

-   -   1. Add 1.6 uL DMSO to 968.4 uL media and then add 30 uL sterile        water    -   2. Add 30 uL sterile water to 970 uL media

For Blanks

-   -   1. Add 30 uL of sample (B1-3) to 970 uL media (final        concentration is 4 uM)    -   2. Add 15 uL of sample (B1-3) to 970 uL media and 15 uL water        (final concentration is 2 uM)    -   3. Add 7.5 uL of sample (B1-3) to 970 uL media and 22.5 uL water        (final concentration is 1 uM)

Aspirate media on 96 well plate and dose with 100 uL of each solution intriplicate followed by detection with a 96 well plate reader, absorbance490 nm.

As shown in FIG. 20, after a 48 hour incubation period, the 5 wt % SN38encapsulated nanoparticles samples, and the SN38 in DMSO/water sample,resulted in reduced cell viability (approximately 25% to 45%, comparedto 100% for the control). In contrast, the blank samples (i.e. thosecontaining the polymeric constituents of the nanoparticles but not theSN38) resulted in 100% cell viability, within error margins.

1. Particles comprising both a branched polymer and a block copolymer.2. Particles as claimed in claim 1 wherein the block copolymer is adiblock copolymer.
 3. Particles as claimed in claim 1 wherein the blockcopolymer comprises a vinyl polymer block.
 4. Particles as claimed inclaim 1 wherein the branched polymer is a branched vinyl polymer. 5.Particles as claimed in claim 1 wherein the branched polymer is abranched vinyl polymer and wherein the block copolymer comprises a vinylpolymer block.
 6. Particles as claimed in claim 1 prepared byco-precipitation.
 7. Particles as claimed in claim 1 wherein thebranched polymer is a branched vinyl polymer, wherein the blockcopolymer comprises a vinyl polymer block, and wherein the particles areprepared by co-precipitation.
 8. Particles as claimed in claim 3 whereinthe vinyl polymer block comprises HPMA, nBuMA, tBuMA, DEAEMA or styrene.9. Particles as claimed in claim 1 wherein the block copolymer comprisesa PEG block.
 10. Particles as claimed in claim 1 wherein the branchedvinyl polymer comprises one or more of the monomers HPMA, nBuMA, tBuMA,styrene, and DEAEMA, and/or wherein the branched vinyl polymer comprisesa brancher selected from EGDMA and BDME.
 11. Particles as claimed inclaim 1 wherein the branched polymer is a branched vinyl polymercomprising: HPMA and EGDMA; or HPMA, nBuMA and EGDMA; or HPMA, tBuMA andEGDMA; or nBuMA and EGDMA; and the block copolymer is a diblockcopolymer comprising HPMA and PEO.
 12. Particles as claimed in claim 1further comprising a drug, prodrug or other biologically activecomponent.
 13. Particles as claimed in claim 12 wherein the drug is anHIV antiretroviral, anticancer drug, or ibuprofen.
 14. Particles asclaimed in claim 1 which are nanoparticles.
 15. Particles as claimed inclaim 1 in solid form.
 16. A composition containing particles as claimedin claim 1 dispersed in water or an aqueous phase.
 17. A method of drugdelivery comprising administering a therapeutically effective amount ofthe particles of claim 12 to a patient in need thereof.
 18. A method oftreating HIV or cancer comprising administering a therapeuticallyeffective amount of the particles of claim 12 to a patient in needthereof.
 19. A method of preparing particles as defined in claim 1comprising: dissolving the branched polymer and block copolymer, andoptionally other component(s), in a solvent to form a solution; addingsaid solution to a different liquid; and removing said solvent to form adispersion of co-precipitated particles.
 20. A method as claimed inclaim 19 further comprising the following subsequent steps, one or moretimes: adding further solution (of the branched polymer and the blockcopolymer, and optionally the other component(s), in the solvent) to theliquid; and removing the solvent.